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Current Protocols in Pharmacology Dec 2019The ryanodine receptor (RyR) is a Ca release channel that is present in the sarcoplasmic reticulum and endoplasmic reticulum (ER) and that plays a central role in...
The ryanodine receptor (RyR) is a Ca release channel that is present in the sarcoplasmic reticulum and endoplasmic reticulum (ER) and that plays a central role in excitation-contraction coupling in skeletal and cardiac muscle. Hyperactivation of RyR by genetic mutations or posttranslational modification can cause various skeletal muscle and arrhythmogenic heart diseases. Inhibitors of RyR are therefore expected to be potential drugs for treatment of such diseases. This article describes assays to evaluate RyR channel activity, including an ER Ca measurement assay that is compatible with high-throughput screening and a [ H]-ryanodine binding assay that provides a quantitative measure of RyR channel activity as a second screen for compound hits. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: [Ca ] assay for ryanodine receptor (RyR) channel activity Support Protocol 1: Determination of dose dependence and isoform selectivity of RyR inhibitors using [Ca ] assay Basic Protocol 2: [ H]-Ryanodine binding assay for RyR channel activity Support Protocol 2: Isolation of microsomes from RyR-expressing HEK293 cells.
Topics: Biological Assay; Calcium; Calcium Channel Blockers; Drug Discovery; HEK293 Cells; Heart Diseases; Humans; Microsomes; Muscle, Skeletal; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 31834676
DOI: 10.1002/cpph.71 -
Journal of Cardiovascular Pharmacology May 2022Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We...
Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca2+ release from the lysosomal Ca2+ stores. Propofol-elicited Ca2+ release was suppressed by 100 μM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca2+ release but abolished the Ca2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca2+ entry elicited by CPA. Ca2+ clearance during CPA-induced Ca2+ discharge was unaffected by a low Na+ (50 mM) extracellular solution, but strongly suppressed by 5 mM La3+ (an inhibitor of plasmalemmal Ca2+ pump), suggesting Ca2+ extrusion was predominantly through the plasmalemmal Ca2+ pump. Propofol mimicked the effect of La3+ in suppressing Ca2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca2+ release and repressed Ca2+ clearance, causing a sustained cytosolic [Ca2+]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.
Topics: Adenosine Triphosphate; Bradykinin; Calcium; Endothelial Cells; Humans; Pain; Propofol; Reactive Oxygen Species; Ryanodine; Ryanodine Receptor Calcium Release Channel
PubMed: 35239284
DOI: 10.1097/FJC.0000000000001246 -
The Journal of Biological Chemistry Oct 1989The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum...
The subunit structure of the rabbit skeletal muscle ryanodine receptor-Ca2+ release channel complex was examined following solubilization of heavy sarcoplasmic reticulum membranes in two zwitterionic detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (Chaps) and Zwittergent 3-14. High and low affinity [3H]ryanodine binding was retained upon solubilization of the complex in Chaps but was lost in Zwittergent 3-14. The purified complex migrated as a single peak with an apparent sedimentation coefficient of approximately 30 and approximately 9 S upon density gradient centrifugation and with isoelectric points of 3.7 and 3.9 upon two-dimensional gel electrophoresis in Chaps and Zwittergent 3-14, respectively. Electron microscopy of negatively stained samples indicated that the distinct four-leaf clover structure of the ryanodine receptor observed in Chaps disappeared following Zwittergent treatment of the 30 S complex and instead showed smaller, round particles. Ferguson plot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial and fully cross-linked and incompletely denatured complexes suggested a stoichiometry of four Mr approximately 400,000 peptides/30 S ryanodine receptor oligomer. [3H]Ryanodine binding to the membrane-bound receptor in 50 microM--1 mM free Ca2+ revealed the presence of both high affinity (KD = 8 nM, Hill coefficient (nH) = 0.9) and low affinity (nH approximately 0.45) sites with a ratio of 1:3. Reduction in free Ca2+ to less than or equal to 0.1 microM or trypsin digestion of the membranes resulted in loss of high affinity but not low affinity ryanodine binding (Hill KD = 5,000 nM, nH = 0.9). Addition of 20 mM caffeine to the nanomolar Ca2+ medium decreased the Hill KD to 1,000 nM without changing the Hill coefficient. Occupation of the low affinity sites altered the rate of [3H]ryanodine dissociation from the high affinity sites. Single channel recordings of the purified ryanodine receptor channel incorporated into planar lipid bilayers also indicated the existence of high and low affinity sites for ryanodine, occupation of which resulted in formation of a subconducting and completely closed state of the channel, respectively. These results are compatible with a subunit structural model of the 30 S ryanodine receptor-Ca2+ release channel complex which comprises a homotetramer of negatively charged and allosterically coupled polypeptides of Mr approximately 400,000.
Topics: Animals; Calcium; Calcium Channels; Cell Membrane; Centrifugation, Density Gradient; Cross-Linking Reagents; Glutaral; Lipid Bilayers; Macromolecular Substances; Membrane Proteins; Microscopy, Electron; Muscles; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum
PubMed: 2550460
DOI: No ID Found -
The American Journal of Physiology Sep 1987Ryanodine affects excitation-contraction coupling in skeletal and cardiac muscle by specifically interacting with the sarcoplasmic reticulum (SR) Ca2+ release channel.... (Comparative Study)
Comparative Study
Ryanodine affects excitation-contraction coupling in skeletal and cardiac muscle by specifically interacting with the sarcoplasmic reticulum (SR) Ca2+ release channel. The effect of the drug at the single channel level was studied by incorporating skeletal and cardiac SR vesicles into planar lipid bilayers. The two channels were activated by micromolar free Ca2+ and millimolar ATP and inhibited by Mg2+ and ruthenium red. Addition of micromolar concentrations of ryanodine decreased about twofold the unit conductance of the Ca2+- and ATP-activated skeletal and cardiac channels. A second effect of ryanodine was to increase the open probability (Po) of the channels in such a way that Po was close to unity under a variety of activating and inactivating conditions. The effects of ryanodine were long lasting in that removal of ryanodine by perfusion did not return the channels into their fully conducting state.
Topics: Alkaloids; Animals; Calcium; Dogs; Electric Conductivity; Ion Channels; Muscles; Myocardium; Ryanodine
PubMed: 2443015
DOI: 10.1152/ajpcell.1987.253.3.C364 -
Journal of Molecular and Cellular... Oct 1995The effects of ryanodine on left ventricular (LV) function and hemodynamics were studied in 16 conscious dogs, chronically instrumented for measurements of LV pressures...
The effects of ryanodine on left ventricular (LV) function and hemodynamics were studied in 16 conscious dogs, chronically instrumented for measurements of LV pressures and dimensions. Systemic infusion of ryanodine (0.5-4 micrograms/kg i.v.) resulted in a dose-dependent depression of cardiac contraction. For example, ryanodine, 4 micrograms/kg i.v., decreased LV fractional shortening by 30.5 +/- 4.1%, LV dP/dt by 41.5 +/- 4.0% and Vcfc by 37.8 +/- 4.1%, while increasing the isovolumic relaxation time constant, tau, from 23.1 +/- 1.4 to 34.1 +/- 3.6 ms without a major effect on preload or afterload. Ryanodine also depressed (P < 0.05) the plateau phase of the mechanical restitution and post-extrasystolic potentiation responses, indicating a direct effect on excitation-contraction coupling. The heart rate dependent positive staircase ("Treppe") was significantly enhanced (P < 0.05) after ryanodine infusion, i.e. LV dP/dt rose by 43.1 +/- 4.7% with an increase in heart rate from 150 to 240 beats/min in the presence of ryanodine 4 micrograms/kg, but by only 7.5 +/- 2.1% without ryanodine. The more pronounced "Treppe" in the conscious dog under the condition of impaired SR calcium release caused by ryanodine, supports the concept that the classical Bowditch "Treppe" reflects either a state of myocardial depression due to alteration in SR calcium handling, or enhanced availability of trans-sarcolemmal Ca2+ influx. This finding may help to understand the discrepancy in the importance of the "Treppe" between conscious animals and more isolated preparations.
Topics: Animals; Calcium Channel Blockers; Cardiac Pacing, Artificial; Consciousness; Depression, Chemical; Dogs; Heart; Heart Rate; Hemodynamics; Myocardial Contraction; Ryanodine; Ventricular Function, Left
PubMed: 8576928
DOI: 10.1016/s0022-2828(95)91227-4 -
Biochemical Pharmacology Aug 1992The effects of ryanodine, a plant alkaloid which alters Ca2+ sequestration in the liver, on O2 uptake and gluconeogenesis were measured. Ryanodine administration to...
The effects of ryanodine, a plant alkaloid which alters Ca2+ sequestration in the liver, on O2 uptake and gluconeogenesis were measured. Ryanodine administration to perfused rat liver resulted in the stimulation of O2 uptake and of gluconeogenesis. Because ryanodine does not affect directly mitochondrial respiration, its stimulatory effect on O2 uptake in the whole cell is likely to be secondary to the increased cytosolic free Ca2+ levels.
Topics: Animals; Gluconeogenesis; Glucose; Liver; Male; Mitochondria, Liver; Oxygen Consumption; Rats; Rats, Inbred Strains; Ryanodine
PubMed: 1510693
DOI: 10.1016/0006-2952(92)90430-q -
Chemical & Pharmaceutical Bulletin Jul 2016Ryanodine (1) is a plant-derived natural product with powerful pharmacological and insecticidal action, and is a potent modulator of intracellular calcium release...
Ryanodine (1) is a plant-derived natural product with powerful pharmacological and insecticidal action, and is a potent modulator of intracellular calcium release channels. Compound 1 possesses a 1H-pyrrole-2-carboxylate ester at the C3-position of heptahydroxylated terpenoid ryanodol (2). Whereas 2 was readily obtained from 1 by basic hydrolysis, 1 has never been synthesized from 2, due to the extreme difficulty in selectively introducing the bulky pyrrole moiety at the severely hindered C3-hydroxyl group of heptaol 2. Here we report chemical conversion of 2 to 1 for the first time. The derivatization was realized through the use of a new protective group strategy and the application of on-site construction of the pyrrole-2-carboxylate ester from the glycine ester and 1,3-bis(dimethylamino)allylium tetrafluoroborate.
Topics: Molecular Structure; Ryanodine
PubMed: 27010543
DOI: 10.1248/cpb.c16-00214 -
Circulation Research Mar 1991Calcium current in mammalian ventricular muscle is altered in the presence of ryanodine. Previous studies performed on rat ventricular cells have shown a slowing of Ca2+...
Calcium current in mammalian ventricular muscle is altered in the presence of ryanodine. Previous studies performed on rat ventricular cells have shown a slowing of Ca2+ current inactivation and suggest the hypothesis that ryanodine, by reducing the release of Ca2+ from the sarcoplasmic reticulum, reduces the availability of Ca2+ for inactivation of Ca2+ current (Ca(2+)-dependent inactivation). Another hypothesis is that the effects of ryanodine on Ca2+ current are due to a mechanical connection of the ryanodine receptor with the L-type Ca2+ channel. To further test these hypotheses we examined the effect of ryanodine on Ca2+ current in single voltage-clamped guinea pig ventricular myocytes that contained Ca2+ indicator and Ca2+ buffer. We used fura 2 (pentapotassium salt) to confirm that the ryanodine we used was capable of abolishing Ca2+ release from the sarcoplasmic reticulum during the period in which it was present. We perfused the cells with 10 mM EGTA to block changes in intracellular Ca2+ concentration. In the absence of internal EGTA, Ca2+ currents displayed biexponential inactivation and Ca(2+)-dependent inactivation (steady-state inactivation curves turned up at positive potentials). Inactivation was slowed by ryanodine at 10 microM. In cells perfused internally with EGTA, however, ryanodine had no effects, and steady-state inactivation curves were not shifted to the right. We conclude that, in guinea pig ventricular myocytes, the effects of ryanodine on Ca2+ current are mediated by Ca2+ and thus the effects of ryanodine do not provide a basis on which to postulate a physical connection between the L-type Ca2+ channel and the ryanodine receptor (sarcoplasmic reticulum Ca2+ release channel).
Topics: Animals; Buffers; Calcium; Egtazic Acid; Electric Conductivity; Guinea Pigs; Myocardium; Osmolar Concentration; Ryanodine; Sodium; Solutions
PubMed: 1742874
DOI: 10.1161/01.res.68.3.897 -
Biophysical Journal Apr 2017Ryanodine (Ryd) irreversibly targets ryanodine receptors (RyRs), a family of intracellular calcium release channels essential for many cellular processes ranging from...
Ryanodine (Ryd) irreversibly targets ryanodine receptors (RyRs), a family of intracellular calcium release channels essential for many cellular processes ranging from muscle contraction to learning and memory. Little is known of the atomistic details about how Ryd binds to RyRs. In this study, we used all-atom molecular dynamics simulations with both enhanced and bidirectional sampling to gain direct insights into how Ryd interacts with major residues in RyRs that were experimentally determined to be critical for its binding. We found that the pyrrolic ring of Ryd displays preference for the RAGGG-F residues in the cavity of RyR1, which explain the effects of the corresponding mutations in RyR2 in experiments. Particularly, the mutant Q4933A (or Q4863A in RyR2) critical for both the gating and Ryd binding not only has significantly less interaction with Ryd than the wild-type, but also yields more space for Ryd and water molecules in the cavity. These results describe clear binding modes of Ryd in the RyR cavity and offer structural mechanisms explaining functional data collected on RyR blockade.
Topics: Animals; Binding Sites; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Protein Structure, Secondary; Ryanodine; Ryanodine Receptor Calcium Release Channel; Thermodynamics; Water
PubMed: 28445755
DOI: 10.1016/j.bpj.2017.03.014 -
Biochemical and Biophysical Research... Apr 1985[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle....
[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca2+-induced Ca2+ release from SR vesicles, inhibits [3H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg2+, Ba2+, Cd2+ and La3+ selectively inhibit the skeletal preparation. The toxicological relevance of the [3H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca2+-ryanodine receptor complexes that may play a role in excitation-contraction coupling.
Topics: Animals; Calcium; Kinetics; Muscles; Myocardium; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum
PubMed: 3985981
DOI: 10.1016/0006-291x(85)91699-7